Animals
The experimental procedures in this study were approved by the Ethics Committee for Animal Experimentation of the Fourth Military Medical University, China. Male Sprague–Dawley (SD) rats, with a weight of approximately 280–320 g, were provided by the Experimental Animal Center of the Fourth Military Medical University. Animals were housed under environmental controlled conditions, with a 12-hour light/dark cycle, an ambient temperature of 24 ± 2°C and free access to food and water. We made efforts to minimize the number of animals used and their suffering.
Experiment design
Experiment I
The objective of this experiment was to examine localization of hemopexin in normal rat brain and to explore its protein level changes in the ischemic penumbra following ischemia-reperfusion. The rats were randomly assigned to the group subjected to middle cerebral artery occlusion (MCAO) or a sham group. Rats in the MCAO group experienced ischemia for 2 h. Next, at 2 h, 6 h, 12 h and 24 h after reperfusion (n=4 rats per group at each time point), the frequency of HPX positive cells in brain sections and the levels of HPX protein in brain homogenates were assessed by immunohistochemistry and Western immunoblotting, respectively.
Experiment II
The objective of this experiment was to evaluate the neuroprotective effects of varying doses of HPX on transient focal cerebral ischemia following intracerebroventricular injection. In this experiment, 50 rats were randomly assigned to 5 groups: control group; vehicle group (intracerebroventricularly injected with 0.1% sodium azide); and dose-dependently delivered HPX injection groups, in which rats received an intracerebroventricular injection of HPX at the following doses: 0.46 mg/ml (low dose), 0.93 mg/ml (middle dose) and 1.86 mg/ml (high dose). Animals were dose-dependently exposed to 5 μl of rat hemopexin reference serum (ICL, Portland, OR, USA) or the carrier vehicle (5 μl, 0.1% sodium azide) at the beginning of reperfusion using a single intracerebroventricular injection following a previously published protocol [11]. Immediately prior to injection, HPX was dissolved in 0.1% sodium azide and diluted to a final concentration in normal saline.
Experiment III
The objective of this experiment was to assess the long-term therapeutic effects of HPX in the setting of transient focal cerebral ischemia. In this experiment, 16 rats were randomly selected into a group receiving the carrier vehicle alone, or a group receiving the test agent HPX (n=8 rats per group). All animals were subjected to MCAO. Both HPX (1.86 mg/ml, 5 μl) and the carrier vehicle (0.1% sodium azide, 5 μl) were administered to the animals by the intracerebroventricular route as soon as the reperfusion procedure had been initiated. A qualified observer, who was blind to the grouping information, evaluated the neurological behavior of the rats daily. These evaluations were measured after MCAO until day seven after administration of HPX, and done according to an 18-point scoring system [12]. Following decapitation of the rats, the brain infarct volumes were assessed.
Western immunoblot analysis
Rats (n = 4 per group, per time-point) were deeply anesthetized before decapitated, and brain tissues quickly removed from the ipsilateral peri-ischemic cortex (ischemic penumbra) in alignment with the corresponding time-points after ischemia-reperfusion and frozen for subsequent tissue homogenization. Protein extracts were obtained by grinding tissue in RIPA buffer containing protease inhibitors. An equal amount of protein (40 μg) was loaded into each lane of a polyacrylamide-SDS gel, subjected to electrophoresis and resolved proteins transferred to a PVDF membrane. Membranes were blocked in 5% BSA and incubated with the appropriate primary antibodies overnight at 4°C. The primary antibodies used in this procedure were; anti-HPX (1:500 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and an anti-rabbit β-actin antibody (1:1000 dilution, CWBIO, Peking, China), which was used as an internal protein loading control. The membranes were washed three times in TBST (Tris-Buffered Saline Tween-20) for 5 min per each, then incubated for 1 h at room temperature with the appropriate goat anti-rabbit secondary antibodies (1:1000 dilution, CWBIO, Peking, China). To visualize specific protein bands, the immunoblots were immersed in enhanced chemiluminescent (ECL) reagent, followed by exposure to ECL- Hyperfilm (Amersham Biosciences, Piscataway, NJ, USA).
Immunofluorescence staining
Animals were first anesthetized with an intraperitoneal administration of 40 mg kg− 1 pentobarbital sodium, then transcardially perfused with saline followed immediately by 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4). Subsequently the brains were removed and immersed in 20% sucrose in phosphate-buffered saline (PBS) overnight at 4°C. Brains were then transferred to a 30% sucrose solution for 24 h. Corneal slices were prepared as 12 um sections using a standard cryostat, and stored at −20°C. Sections were blocked in 10% normal goat serum supplemented with 0.3% Triton X-100 for 1 h at room temperature. Sections were next incubated with a combination of primary anti-mouse glial fibrillary acidic protein (GFAP, at a dilution of 1:500; Sigma-Aldrich, Madrid, Spain) and a primary rabbit polyclonal antibody directed against HPX (at a dilution of 1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C overnight. Sections were next washed three times in PBS, then incubated in NeuroTrace (at a dilution of 1:1000; Molecular Probes, Eugene, OR, USA) or with two secondary antibodies for 2 h at room temperature. Secondary antibodies were: donkey anti-mouse conjugated to green-fluorescent Alexa Fluor 488 (at a concentration of 1:500; Vector Laboratories, Inc., Burlingame, CA) and donkey anti-rabbit conjugated to red-fluorescent Alexa Fluor 594 (at a dilution of 1:500; Vector Laboratories). Sections were incubated with DAPI (1 ng/μl; Sigma-Aldrich, Poole, UK) to stain the nuclei for 5 min at room temperature. Fluorescent signals were detected by confocal laser scanning microscopy (Olympus, FV1000).
Focal cerebral ischemia
Focal cerebral ischemia was induced in rats by MCAO using an intraluminal filament technique, as previously described [13, 14]. Briefly, rats were fasted for 12 h prior to surgery but were allowed free access to water. Anesthesia was induced by intraperitoneal injection of pentobarbital sodium (40 mg/kg in normal saline). Next, the right common carotid artery (CCA) and the right external carotid artery (ECA) were exposed through a ventral midline neck incision, and were ligated proximally. A 4/0 monofilament nylon suture (Beijing Sunbio Biotech Co., Ltd.) was inserted through an arteriectomy in the common carotid artery just below the carotid bifurcation and into the internal carotid artery approximately 18–20 mm distal to the carotid bifurcation until mild resistance was felt [15]. Next, the middle cerebral artery was occluded. After 2 h of ischemia, the reperfusion process was accomplished by withdrawing the suture then reapplying the suture to the wound. During this process cerebral blood flow was monitored through a disposable fiber optic probe (with a diameter of 0.5 mm) connected to a laser Doppler unit (PeriFlux 5000, Perimed AB, Stockholm, Sweden). Rats that showed more than a 70% reduction in cerebral blood flow were retained in their corresponding groups for data recording (Figure 1). Body temperature of the rats was maintained at 37 ± 0.5°C during the procedure.
Intracerebroventricular injection
Anesthetized rats were placed on a stereotaxic apparatus and four stainless-steel screws were secured to the skull and occluded. An incision to the scalp exposed the surface of the skull and bregma. A burr hole was drilled into the bone of the right hemisphere with a stainless steel 26-gauge cannula, located 1.5 mm lateral to, and 0.8 mm posterior to the bregma. A 5 μl Hamilton syringe was introduced slowly to 3.5 mm beneath the dural surface to allow dose dependent exposure of rats to 5 μl of rat hemopexin reference serum HPX (ICL, Portland, OR) or vehicle (0.1% sodium azide) by injection. The injection needle was maintained in situ for 5 min before withdrawal.
Neurobehavioral evaluation and assessment of infarct
At 24 h (or 2, 3, 4, 5, 6 and 7d) after reperfusion, the neurologic behavior of the rats was determined by a qualified observer blinded to the rat grouping strategy, and according to a previously described 18-point scoring system [12]. Rats were anesthetized, decapitated, and the brains were removed, sliced into six separate 2 mm thick coronal sections and stained with a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, St. Louis, MO) at 37°C for 10 min [13, 14]. The slices were then fixed in 4% formalin prior to digital photographic images being recorded (Canon, Tokyo, Japan). Unstained areas were defined as infarcts and were measured by image analysis software (Adobe Photoshop, Version CS3). To exclude the effect of ischemia-induced cerebral edema on the infarct size, the percent of the infarct volume was calculated indirectly according to the formula: ([total contralateral hemispheric volume] - [total ipsilateral hemispheric stained volume]) / (total contralateral hemispheric volume) × 100.
Statistical analyses
Statistical analyses were made using the SPSS 14.0 data analysis software program. All values, with the exception of the neurologic scores, were described as mean ± SEM, and were analyzed by one-way analysis of variance (ANOVA). Differences between two groups were detected by applying Tukey’s post-hoc test. The neurological scores were described as the median (interquartile range), and these data sets were compared by applying the Kruskal-Wallis test followed by the Mann–Whitney U-test and Bonferroni’s post-hoc correction. Statistical significance was defined as attaining at least a value of P< 0.05.